Journal of Medicinal Research Vol. 5(9), pp. 1526-1530, 4 May, 2011 Available online at http://www.academicjournals.org/JMPR ISSN 1996-0875 ©2011 Academic Journals

Full Length Research Paper

Assay of flavonoid aglycones with HPLC in four species of genus

Zeb Saddiqe 1*, Ismat Naeem 2, Alya Maimoona 1, Asmita V. Patel 3 and Claire Hellio 4

1Department of Botany, Lahore College for Women University, Lahore, Pakistan. 2Department of Chemistry, Lahore College for Women University, Lahore, Pakistan. 3School of Pharmacy and Biomedical Sciences, University of Portsmouth, UK. 4School of Biological Sciences, University of Portsmouth, UK.

Accepted 1 July, 2010

Hypericum plants are widely used in phytotherapy in many countries. A number of plants of this genus have exhibited various pharmacological activities attributed to their flavonoid content. The present paper describes a reverse-phase HPLC method developed for the assay of 7 flavonoid aglycones (, , , , , luteolin and apigenin) in polar extracts of four Hypericum species from UK, , L., , Arechav, Hypericum x moserianum ( x ) Andrê and , L. The identity of detected flavonoids was confirmed by comparing their retention times with those of corresponding standards. The content of detected flavonoids varied from 816.69 (mg/Kg fresh wt.) in Hypericum moserianum to 6726.52 (mg/Kg fresh wt.) in Hypericum olympicum . Quercetin and apigenin were present in all the species while myricetin and kaempferol were detected only in H. moserianum .

Key words: Hypericum andorsaemum , Hypericum ericoides , Hypericum x moserianum , Hypericum olympicum , flavonoids, quercetin, HPLC.

INTRODUCTION

The importance of medicinal plants to the health of for the last two hundred years (Dias et al., 1998). The individuals and communities is known since antiquity. whole extract has antidepressive effects on The medicinal value of these plants lies in some neurotransmitter levels in the brain (Bloomfield et al., chemical substances that produce a definite 1996). The antidepressive (Kumar et al., 1999), physiological effect on human body. The most important anticarcinogenic (Zeng et al., 2006) and antimicrobial of these bioactive constituents of plants are alkaloids, (Sokeman et al., 1999) activities of these plants are tannins, phenolic compounds and flavonoids (Hill, currently under investigation. The increasing interest in 1952). Among these, flavonoids are the most ubiquitous the chemistry of this genus has led to the isolation of group of plant secondary metabolites demonstrating a more than 100 components with different biological wide range of biochemical and pharmacological effects, activities (Quing-Li et al., 1998; Li-Hong and Keng, including anti-inflammatory (Owoyele et al., 2008), 2000). antibacterial (Hernández et al., 2000), antifungal (Li et Methanolic extract from the aerial parts of Hypericum al., 2005), antioxidant (Bernardi et al., 2007) and plants typically contains hypericins, hyperforins and anticarcinogenic (Seelinger et al., 2008). phenolic compounds (Barnes et al., 2001). It is Hypericum species are medicinal plants known as especially rich in phenolics, caffeic acid, chlorogenic healing herbs due to their various medicinal properties acid, proanthocyanidin, prenylated derivatives of phloroglucinol and flavonoids (Mojca et al., 2005). The pharmacological activity of Hypericum species has been linked mainly to the flavonoids and phloroglucinols *Corresponding author. E-mail: [email protected]. (Decosterd et al., 1991; Rocha et al., 1995; Butterweck Tel: +92-42-99203801-9/245. et al., 2000; Bernardi et al., 2007). Numerous flavonoids Saddiqe et al. 1527

Table 1. Flavonoids isolated from genus Hypericum .

Flavonoid Reference Astraglin Kitanov, 1988 Astilbin Butterweck et al., 2000 3,8”-Biapigenin Kurkin and Pradivtseva, 2007 6,8”-Diquercetin Kurkin and Pradivtseva, 2007 Kitanov, 1988; Makovetskaya, 1999 Isoquercitrin Butterweck et al., 2000 Kaempferol Kitanov et al., 1979; Naeem et al., 2010; Saddique et al. 2011 Kitanov, 1988 Myricetin Kitanov et al.,1979; Naeem et al., 2010; Saddique et al. 2011 Myricetin 3-glucoside Kitanov, 1988 Kitanov, 1988 Quercetin Kitanov et al., 1979; Naeem et al., 2010; Saddique et al. 2011 Kitanov, 1988; Makovetskaya, 1999 Kurkin and Pradivtseva, 2007

Flavonoid R1 R2 R3 R4 R5 Quercetin OH H H OH OH Myricetin OH OH OH OH OH Isorhamnetin OMe OH H OH OH Rhamnetin OH OH H OH OMe Kaempferol H OH H OH OH Luteolin H OH OH H OH Apigenin H OH H H H

Figure 1. Chemical structures of selected flavonoids.

have been identified in various species of genus studied are depicted in Figure 1. Hypericum (Table 1). Today, high performance liquid chromatography is established as the most convenient method which EXPERIMENTAL enables separation and identification of flavonoids using Chemicals and reagents various detection systems (Janeska et al., 2007; Plazoni č et al., 2009). High performance liquid All solvents used were purchased from Merck, Germany and chromatography methods are developed for qualitative were of analytical grade. Chemical standards for quercetin (98%), and quantitative analyses of flavonoids in various plant luteolin (95%), myricetin (95%), rhamnetin (99%), isorhamnetin (95%), kaempferol (96%) and apigenin (95%) were purchased materials (Bobzin et al., 2000; Dubber and Kanfer, from Sigma- Aldrich. The purity of these flavonoids was assumed 2004). The present paper describes a simple, precise, as provided by suppliers and no adjustments were made in the rapid and reproducible method to identify and quantify quantitative analysis of the commercially available products. seven relevant flavonoid aglycones in the methanolic Acetonitrile, methanol and water used for HPLC analysis were extracts of aerial parts of four Hypericum species, HPLC grade purchased from Merck, Germany. Hypericum androsaemum L., Hypericum ericoides Arechav, Hypericum x moserianum Andrê and Plant material Hypericum olympicum L. from UK using HPLC. The methanolic extracts were partitioned further to The four Hypericum species purchased from Perryhill Nurseries determine the solvent most suitable for the extraction of were grown in the green house of University of Portsmouth, UK these flavonoid aglycones. Structures of the aglycones for one year. The aerial parts of the four species were used in the 1528 J. Med. Plant. Res.

Table 2. Percentage yield of the extracts recovered from the four Hypericum species.

Plant species MeOH (%) n-Hexane (%) Chloroform (%) EtOAc (%) Acetone (%) Aqueous (%) H. androsaemum 35.50 0.96 3.41 0.84 0.83 26.10 H. ericoides 22.0 3.00 1.85 0.91 1.40 15.00 H. x moseriarum 20.0 1.52 0.24 0.33 0.50 18.30 H. olympicum 48.5 3.25 2.40 0.93 1.81 36.20

Table 3. Contents of flavonoids (mg/kg fresh weight) in methanolic extracts of the four Hypericum species.

Flavonoid tR (min) H. andorsaemum H. ericoides H. moserianum H. olympicum Quercetin 2.059 405.0 855.0 9.83 48.00 Myricetin 1.787 nd nd 540.00 nd Isorhamnetin 2.740 10.50 13.72 nd 1038.95 Rhamnetin 3.750 383.65 4.25 nd 3622.00 Kaempferol 2.639 nd nd 266.03 nd Luteolin 1.966 nd nd 0.50 4.57 Apigenin 2.487 482.0 0.67 0.33 2013.00 Sum total of flavonoids 1281.15 873.64 816.69 6726.52

tR = retention time, nd = Not Detected.

present study. Herbarium specimens of all the species were 100 g mL -1. All samples were stored at 4°C and were filtered lodged in the Herbarium of Hampshire County Council Museum through a 0.45 m filter before undertaking HPLC analysis. Service, Winchester, Hampshire, UK (Index Herbariorum code HCMS; accession number Bi 2000 16. 370, 371, 372 and 373 for H. androsaemum, H. ericoides, H. x moserianum and H. Apparatus and Conditions olympicum , respectively). Qualitative and quantitative HPLC separation of the flavonoid aglycones in the crude extracts was performed with Waters HPLC Extraction procedure system, equipped with a pump (1500 series), a column oven, a UV detector (2487) and a reversed-phase, pre-packed C18 For the extraction of plant material, modified method of Vict όria et column (250 x 4.6 mm, 5 m particle size). The data was al (2009) was used. The fresh aerial parts of the four Hypericum analyzed and processed using the installed Empower Software. species (500 gm each) were extracted with methanol at room The column was maintained at room temperature. The mobile temperature for 24 h with occasional stirring. The methanolic phase was run at as a flow rate of 1.0 mL/min and consisted of extract was concentrated under reduced pressure in a rotary acetonitrile/water 1:1 acidified with 1% acetic acid. Throughout evaporator to give a gummy residue. The gummy residue was the experiment all injection volumes were 10 l and the dissolved and suspended in water and partitioned between n- compounds were detected at 254 nm. The separated flavonoid hexane, chloroform, ethyl acetate and acetone sequentially (three compounds were initially identified by direct comparison of their times each). These five extracts were condensed down under retention times with those of standards. Standard solution was vacuum. The respective yields were calculated as percentage then added to the sample and peaks were identified by the using the formula: (crude extract weight/plant material weight) x observed increase in their intensity. This procedure was 100 and are shown in Table 2. performed separately for each standard. The flavonoid content was calculated from the peak areas of HPLC chromatograms from the 3 replicate samples. HPLC analysis

Preparation of standard solutions RESULTS AND DISCUSSION Standard stock solutions of the reference compounds were prepared in HPLC grade methanol at a concentration of 100 g The result of qualitative and quantitative analysis of mL -1 and stored in a refrigerator at -20°C until use. All standard flavonoids in the four Hyepricum species is shown in solutions were filtered through 0.45 m filters and diluted as Table 3. There are not much data on the phytochemical necessary with methanol. analysis of the four Hypericum species studied. This

study is the first report on the flavonoid content of these

species using HPLC as the chromatographic technique Sample preparation and shows their valuable chemical composition For HPLC analysis a weighed amount of each extract was justifying their use in traditional medicine. The species dissolved in HPLC grade methanol to give a concentration of H. olympicum has been shown to be richer in variety as Saddiqe et al. 1529

Table 4. Quantity of flavonoids (mg/kg fresh weight) recovered from the different extracts of the four Hypericum species after partitioning.

Flavonoids H. andorsaemum H. ericoides H. moserianum H. olympicum EA AC AQ EA AC AQ EA AC AQ EA AC AQ Quercetin nd nd 403.00 nd nd 852.0 nd 9.65 nd 47.42 nd nd Myricetin nd nd nd nd nd nd nd nd 538.01 nd nd nd Isorhamnetin 10.01 nd nd nd 13.63 nd nd nd nd nd 3.50 1035.30 Rhamnetin 10.74 5.12 366.00 nd 4.10 nd nd nd nd nd 0.062 3620.00 Kaempferol nd nd nd nd nd nd 0.55 nd 264.12 nd nd nd Luteolin nd nd nd nd nd nd 0.45 nd nd nd 4.45 nd Apigenin nd nd 476.00 0.61 nd nd 0.29 nd nd 30.00 nd 1982.00

EA = ethyl acetate fraction, AC = acetone fraction, AQ = aqueous fraction.

well as in quantity of the flavonoid aglycones studied, seven reference standards obtained under the same ranging from 4.57 (luteolin) to 3622.00 (rhamnetin) conditions. The analysis time is an important factor in (mg/Kg) of fresh plant material. Comparing the analytical work and the run time should be reduced to flavonoid composition of the four species reveals minimum in order to optimize equipment use and greater similarity among H. androsaemum. H. ericoides reduce solvent consumption. The present method gave and H. olympicum having quercetin, isorhamnetin, a quick and accurate method for the separation of rhamnetin and apigenin. Additionally, myricetin and seven flavonoid aglycones with a run time of just 7 min. kaempferol were found only in H. x moserianum indicating the possibility of its chemosystematic distinction. Conclusion To analyze the effect of solvent type on the extraction of flavonoids, the crude methanolic extracts of all the The results showed that the four Hyperiucm species three species were partitioned to give non-polar (n- studied are a rich source of the important biologically hexane and dichloromethane) and polar (ethyl acetate, active flavonoids. The described HPLC procedure could acetone and water) fractions. The polar fractions were be useful for the qualitative and quantitative analysis of analyzed for their flavonoid content following the same flavonoids in the genus Hypericum . The results might procedure as for methanolic extract. The result of be helpful for providing chemotaxonomic relationships flavonoid analysis in these fractions is shown in Table between species of this genus. 4. The result indicated that most of the flavonoids after partitioning were detected in the aqueous fraction indicating the aqueous phase to be the most effective ACKNOWLEDGEMENTS for the isolation of flavonoid aglycones from the plant material. The authors are thankful to Higher Education Flavonoids are important secondary metabolites with Commission (HEC) Pakistan and HEC-BC Link for widespread occurrence in plant kingdom. A number of providing funding and lab facilities for this research flavonoids have shown to possess antibacterial work. (Harborne, 2000), anticarcinogenic (Neuhouser et al., 2004) and antiviral (Ma et al., 2001; Meragelman et al., 2001) activities. REFERENCES

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